Structure and method for releasing substance therefrom

ABSTRACT

A structure comprises at least a porous body holding a substance releasably, comprising a capping member for keeping the substance inside the pore and/or on at least a part of the entire surface of the porous body, and a connecting member for connecting the porous body and the capping member separably, the connecting member comprising a biopolymer compound. 
     A method for releasing a substance from the structure set forth comprises the steps of applying stimulation from outside to the structure, and cleaving at least one of the bonding between the connecting member and the capping member and the bonding between the connecting member and the porous member to make the substance releasable from the structure.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a structure comprising a porous body, acapping member, and a connecting member for connecting the porous bodyand the capping member, and relates also to a method for releasing asubstance kept by the capping member in the porous body.

2. Related Background Art

Many investigations are being made on stimulation-responsive materialswhich function by changing a shape or a property thereof in response toan external stimulation such as light irradiation, electric fieldapplication, and chemical substance addition; or an environmental changesuch as a temperature change, and a pH change. The function of thestimulation-responsive material can be controlled from outside byutilizing the property, and is promising in various application fields,such as drug delivery.

Generally, a drug is dosed into a body by injection, oraladministration, painting, or a like method, and the dosed drugcirculates in the body to reach a target site. In these dosing methodgenerally, the drug can diffuse to a portion other than the targeteddiseased portion or may be absorbed or decomposed in a digestive tractor other tract undesirably, reaching the diseased portion finally in aquantity (concentration) much smaller than the dosed quantity.Therefore, the drug is dosed in a quantity larger than that actuallyrequired.

To solve the above problem, various measures are taken, includingmodification of a portion of the drug compound relating to theabsorption or decomposition without lowering the drug effect, use of adrug carrier, and so forth. The drug carrier carries a drug selectivelyto a targeted diseased portion or an objective matter such as internalorgans, tissues, cells, and pathogens. This technique employing a drugcarrier is called a drug delivery system (hereinafter referred to as“DDS”). This DDS technique can increase the treatment effect of thedrug, and can decrease the drug dose to lower the adverse effect of thedrug advantageously. As the drug carrier, liposomes, lipid microsheres,and the like are being investigated.

A liposome is a spherical vesicle of lipid having a hydrophilic portionand a hydrophobic portion, constituted of a bilayer with the hydrophobicgroups placed inside to be stable to an outside water environment. Aliposome having a stable bilayer structure can be formed by dissolving anatural lipid such as lecithin and cholesterol in an organic solvent anddispersing it in water by ultrasonic treatment or a like method. In theliposome forming process, a drug to be carried can be enclosed in theliposome. A drug which is a hydrophobic substance is held in the insideof the bilayer, whereas a drug which is hydrophilic is held in theinside water phase enclosed by the bilayer.

Lipid microspheres can be prepared by suspending a drug-containingsoybean oil and lecithin in water, having the lecithin on the surfaceand the drug-containing soybean oil enclosed therein. Medicalformulations comprising such lipid microspheres enclosing ananti-inflammatory drug are used in clinical treatment. Japanese PatentApplication Laid-Open No. H05-221852 discloses a method for forminglipid microspheres containing a fatty acid ester and having fineparticle surfaces by dissolving a fat-soluble anticancer agent in thefatty acid and homogenizing the fatty acid ester solution with asurfactant such as a phospholipids. However, the liposome structure canreadily be destroyed by contact with a lipid or protein in blood, beingnot stable in vivo for a long time. For stabilization, modification ofthe liposome by polyethylene glycol (PEG) or a polysaccharide isinvestigated. However, the stability is not satisfactory yet. Further,to use the lipid microsphere, the drug should be fat-soluble, butfat-soluble drug is not stable in the blood similarly as the liposome,and is liable to migrate to reticuloendothelial system. The liposomeneeds to be improved more for use as the drug carrier.

For improving the stability of the aforementioned drug carriers in theblood, a report is presented which utilizes a block copolymerconstituted of polyethylene glycol as the hydrophilic portion andpolyamino acid as the hydrophobic portion to form a micelle-likestructure in an aqueous solution or a phosphate buffer solution (DrugDelivery System, Vol.6(2), pp77, 1991). This block copolymer is capableof forming a micelle-like stable structure in an aqueous solution or anaqueous phosphate buffer solution. A drug like an anti-cancer agent isenclosed in this structure for stabilization in the blood.

U.S. Pat. No. 3,854,480 discloses a drug delivery system which releasesa drug at a controlled rate for a long term. This system employs astructure constituted of a film and a drug-containing core, the filmbeing composed of a polymer such as polyethylene and an ethylene-vinylacetate copolymer, and the drug-containing core being composed of amatrix of polymethylsiloxane or the like containing a drug dispersedtherein. The drug is released to outside from the core through the filmby diffusion.

The above disclosures do not teach a technique for releasing the drugselectively to the targeted diseased portion. For more effective medicaltreatment and lower side effect of the drug, a drug carrier is demandedto be developed which has capability of controlling the drug release ata targeted site as well as the stability after administration in theblood and other portion of the body and on the body surface.

To meet such demand, a technique is disclosed which encloses a drug or achemical compound in a silica type porous material stable in the bodyfluid like blood and releases the compound with control by outsidestimulation (J. Am. Chem. Soc., Vol.125, pp4451, 2003). In the disclosedtechnique, the material for enclosing the drug is mesoporous silica(MCM-41) having an average particle size of 200 nm, and an average porediameter of 2.3 nm (hereinafter referred to as a “silica structure”)modified by 2-(propyldisulfanyl)ethylamine. By this technique, thesilica structure is immersed in an aqueous solution of ATP andvancomycin, and thereto CdS (average particle size: 2.0 nm) is addedwhich has been modified by acetic acid thiol to cause bridging bychemical bonding between the amino groups on the surface of the silicastructure and the carboxyl groups of the CdS surface to cap the silicastructure to enclose the drug. The compound enclosed in the silicastructure is released by treatment with a reducing agent such as DTT andmercaptoethanol to cleave the S-S bond of the disulfanyl groups toremove the CdS from the mesoporous silica.

A report (J. Material. Biomed. Mater. Res. 51, pp293 (2000)) discloses atechnique for destroying nano particles constituted of cores of ahydrogel of an N-isopropylacrylamide-acrylamide copolymer (NIPAAm-AAm)coated with gold by swelling of the NIPAAm-AAm core by absorption ofnear infrared light of 800 to 1200 nm and a resulting photothermalconversion reaction. This report suggests also possibility of drugrelease, by light response, from NIPAAm-AAm containing a drug suspendedor dissolved therein. The near infrared light in the range from 800 to1200 nm penetrates a human tissue but is harmless. However, the corematerial, NIPAAm-AAm, is not completely safe to the tissue of the humanbody.

In the aforementioned methods, the silica structure enclosing a drug anda reducing agent are allowed to coexist, or a gold-coated fineparticulate NIPAAm-AAm is used for the controlled release of the drug.However, the localization of the reducing agent in the diseased portionhas technical problems in the method and the safety to be solved.

SUMMARY OF THE INVENTION

The present invention has been made to satisfy the demand for astructure for controlled release of a drug or a like substance. Thepresent invention intends to provide a structure which is capable ofsurely controlling release of a substance like a drug at a prescribedsite.

After comprehensive investigation to solve the above problems, theinventors of the present invention have found a structure describedbelow.

According to an aspect of the persent invention, there is provided astructure comprising at least a porous body holding a substancereleasably, comprising a capping member for keeping the substance insidethe pore and/or on at least a part of the entire surface of the porousbody, and a connecting member for connecting the porous body and thecapping member separably, the connecting member comprising a biopolymercompound.

The connecting member preferably has a first site for bonding to theporous body and a second site for bonding to the capping member.

The site of the connecting member for bonding to the capping memberpreferably comprises at least a portion of an antibody variable regioncapable of bonding to the capping member.

In the structure, gold is preferably contained at least a part of thesurface of the capping member, and the bonding site of the connectingmember is preferably capable of bonding to the gold. The bonding site ofthe connecting member for bonding to the capping member preferablycomprises one or more amino acid sequence selected from the groupconsisting of amino acid sequences of SEQ ID NO:1 to 57.

The bonding site of the connecting member for bonding to the cappingmember preferably comprises one or more amino acid sequence selectedfrom the group consisting of amino acid sequences derived from the aminoacid sequences SEQ ID NO:1 to 57 by deletion, substitution or additionof one or more amino acids.

In the above structure, the bonding site of the connecting member forbonding to the porous body preferably comprises a peptide capable ofbonding to the porous body. At least a part of the surface of the porousbody preferably contains at least one of metals and metal oxides, andthe bonding site of the connecting member to the porous body ispreferably capable of bonding to the part of the surface of the porousbody containing at least one of metals and metal oxides. Alternatively,at least a part of the surface of the porous body contains siliconoxide, and the bonding site of the connecting member to the porous bodyis preferably capable of bonding to the part containing the siliconoxide. The bonding site of the connecting member for bonding to theporous body comprises two or more amino acid sequences selected from thegroup consisting of

-   (1) amino acid sequence SEQ ID NO:80, and-   (2) amino acid sequences derived by deletion,    substitution or addition of one or more amino acid from or to amino    acid sequence SEQ ID NO:81.

The bonding of the connecting member to at least one of the cappingmember and the porous bodies is preferably broken by externalstimulation to render releasable the substance kept by the cappingmember in the porous body.

According to another aspect of the present invention, there is provideda method for releasing a substance from the structure set forth inabove, comprising the steps of:

applying stimulation from outside to the structure, and cleaving atleast one of the bonding between the connecting member and the cappingmember and the bonding between the connecting member and the porousmember to make the substance releasable from the structure. Thestimulation is preferably a physical action. The physical stimulation ispreferably at least one of light and a magnetic field, and the physicalstimulation causes a change of temperature of the capping member, andthe temperature change cleaves the bonding between the capping memberand the connecting member.

The structure of the present invention is effective as mentioned below.Firstly, in holding one or more substances in the structure, the featureof a porous body having a large surface area per unit volume can beutilized most effectively for holding the substance to be released on atleast a part of the entire surface comprising the outside surface andinside surface of pore wall, preferably at least on the inside surfaceof the pore and the pore opening periphery. Secondly, spontaneousdiffusion of the substance held by the porous body can be prevented by acapping member. Thirdly, the connection of the capping member with theporous body can be stabilized by physical or chemical connection betweenthe porous body and the capping member surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates schematically a structure of the present invention,and release of a compound from the structure.

FIGS. 2A, 2B, 2C, 2D, 2E, and 2F illustrate examples of preparation of avector for production of a biopolymer compound.

FIGS. 3A, 3B, and 3C illustrate examples of preparation of a vector forproduction of a biopolymer compound.

FIGS. 4A, 4B, 4C and 4D illustrate schematically a constitution of agold-bonding protein.

FIG. 5 illustrates schematically a constitution of a gold-bondingprotein.

FIG. 6 illustrates schematically a constitution of a gold-bondingprotein.

FIG. 7 illustrates schematically a constitution of a gold-bondingprotein.

FIG. 8 illustrates schematically a constitution of a gold-bondingprotein.

FIG. 9 illustrates schematically a constitution of a gold-bondingprotein.

FIG. 10 illustrates schematically a constitution of a gold-bondingprotein.

FIG. 11 illustrates schematically a constitution of a gold-bondingprotein.

FIG. 12 illustrates schematically a constitution of a gold-bondingprotein.

FIG. 13 illustrates schematically a constitution of a gold-bondingprotein.

FIGS. 14A, 14B, 14C, 14D, 14E, 14F, 14G, and 14H illustrate respectivelya fine structure: a porous structure, an opal structure, a reversed opalstructure, a columnar structure, a convexed structure, a concavedstructure, a projecting structure, and a fibrous structure.

DETAIL DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred embodiments of the present invention are explained belowin detail by reference to drawings. (Incidentally, the embodiments notexperimentally practiced are described later.)

(Structure)

The structure of the present invention holds one or more substances tobe releasable. The structure comprises a porous body, a capping member,and a connecting member for connecting the porous body and the cappingmember physically or chemically, the connecting member comprising anorganic compound comprising a biopolymer compound.

The size of the structure depends on the size of the porous body as thebase material described later, and can be selected and designed for theuse of the structure. For example, for use for DDS, the diameter ormaximum length of the particle is preferably not more than 200 nm; fordelivery to finer blood capillary, preferably not more than 50 nm. Thelower limit of the size is not specially limited, but the diameter ormaximum length of the particle is not less than 5 nm. The releasablesubstance is held by at least one of an outside surface of the porousbody, an inside space of the pores, and an inside surface of the porousbody, namely an inside wall of the pore. The substance is heldpreferably on the outside surface or the inside surface, more preferablyat least on the inside surface and opening periphery of the pore.

(Porous Body)

The porous body for the structure of the present invention has a largesurface area per unit volume, namely a specific surface area, forachieving the effect of the present invention. The pores of the porousbody are open to the outside of the structure. The shape of the pore isselected to be suitable for the substance to be held releasably in thepore and for the outside environment, namely properties of a liquid orsolution suspending or dissolving the substance. The pore preferablypenetrates through the porous body. The diameter of the pore rangespreferably from 1 nm to 10 μm, more preferably from 50 nm to 1000 nm.

The constituting material of the porous body is selected suitably fromknown materials. The material is selected from the group of metals,metal oxides, insoluble inorganic salts, inorganic semiconductors,organic semiconductors, and organic polymer compounds, and compositesthereof; the organic polymer compounds including natural polymercompounds, synthetic polymer compounds, plastics, pulp, woven cloths,and nonwoven cloths; and composites thereof. The metals include gold,silver, platinum, aluminum, and copper. The metal oxides include siliconoxide, alumina, titanium oxide, zinc oxide, magnetite, ferrite, NbTacomposite oxide, WO₃, In₂O₃, InSnO, MoO₃, V₂O₅, and SnO₂. The insolubleinorganic salts include hydroxyapatite, and calcium phosphate gel. Theorganic polymer compounds include polymers and copolymers produced bypolymerizing or copolymerizing a polymerizable monomer or monomersselected from the group of known styrene monomers such as styrene,α-methylstyrene, and β-methylstyrene; methacrylate monomers such asmethyl acrylate, and ethylacrylate; methylene aliphatic monocarboxylicacid esters; vinyl esters such as vinyl acetate, and vinyl propionate;vinyl ethers such as vinyl methyl ether; and vinyl ketones such as vinylmethyl ketone.

Further, the structure may be formed from any of the materials includingfilms constituted of a polymer such as polyethylene terephthalate (PET),diacetate, triacetate, cellophane, celluloid, polycarbonate, polyimide,polyvinyl chloride, polyvinylidene chloride, polyacrylate, polyethylene,polypropylene, and polyesters; porous polymer films of polyvinylchloride, polyvinyl alcohol, acetylcellulose, polycarbonate, nylon,polypropylene, polyethylene, Teflon, and the like; wood plates; glassplates; silicon substrates; cloths produced from cotton, rayon,acrylates, silk, polyesters or the like; and paper sheets of wood-freepaper, medium quality paper, art paper, bond paper, regenerated paper,baryta paper, cast-coated paper, corrugated paper, and resin-coatedpaper, but is not limited thereto.

Among the above materials, metal oxides are preferred as the materialfor the drug carrier in consideration of stability in the body fluid,the biocompatibility, and the production cost of the porous body. Themetal oxides include silicon oxide, aluminum oxide, iron oxide, andferrite. When the release of the material from the structure iscontrolled by light/magnetism, silicon oxide is the most suitable as theporous body constituting material which does not absorb the light or isnot affected by the magnetism.

The porous body may have any constitution shown below.

-   Hollow column construction: many cylindrical hollows of any shape    are arranged (FIG. 14A)-   Porous construction: many pores of any shape are formed at random    (FIG. 14B)-   Opal constitution: spherical matters are closely packed (FIG. 14C)-   Reversed opal constitution: material/pore is reversed in the opal    constitution (FIG. 14D)-   Concave constitution: many concaves are formed in the substrate    (FIG. 14E)

The porous body may be provided on a suitable supporting base plate, ormay be an independent body. The supporting base plate may be made of aknown material including metals, metal oxides, plastics, and compositesthereof.

The porous body may be formed by any working method suitable for thematerial selected as above. The working method includesphotolithography, etching, sand blasting, and FIB working. When aluminumis selected as the material, the porous body can be formed by anelectrochemical method such as anodation. When silicon oxide is selectedas the material, the porous body can be formed by a sol-gel method. Thismethod enables formation of porous body having an average pore diameterranging from 2 to 20 nm, and an average particle diameter of about 200nm. An example of this method is described.

An alkoxysilane solution containing a surfactant is applied under acidicconditions to form a coating layer. The layer is treated at 35° C. for20 hours to allow the reaction to proceed, and is further heated at 80°C. for 48 hours. Thereby a silicon oxide layer is formed which containsa surfactant phase distributed in a net state. Then the surfactant phasedistributed in the layer is eliminated by heating at 500° C. for 6hours. The resulting porous material layer has a porous constitutionhaving pores of the diameter ranging from 1 nm to 1000 nm at the regionof the eliminated surfactant phases. The surfactant may be eliminated,other than the above heating method, by treatment with an organicsolvent. The method is selected to be suitable depending on theproperties, such as heat resistance and solvent resistance, of the baseplate employed.

(Capping member)

The capping member keeps the compound in the structure of the presentinvention not to disappear by spontaneous diffusion or a like process,and releases the compound outside on receiving an external signal suchas light and a magnetic field change. On receiving light or a magneticfield change, the capping member causes preferably a temperature changeat the periphery thereof.

Suitable materials for the capping member includes metals such as gold,silver, copper, platinum, zinc, aluminum, and silicon; magnetizablemetals such as iron, cobalt, and nickel; and oxides thereof. Of these,preferred are gold, silver, copper, and platinum which are capable ofconverting readily the absorbed light or the magnetic field to heat, butare not limited thereto.

The size of the capping member is not limited. However, when the cappingmember is much smaller than the pore diameter of the porous body, thesubstance held by the porous body can diffuse through a gap between thepore wall face and the capping member. On the other hand, when thecapping member is much larger than the porous body, the capping membercovers incompletely the openings of the plural pores not to functionsufficiently as the capping member. In consideration of the abovepreferred pore diameter of 1-10 nm, or considering the diameter of theporous body, the diameter of the capping member ranges preferably from 1to 100 nm, more preferably from 2 to 50 nm.

From the aforementioned reasons, gold is the most suitable as thematerial of the capping member. For example, a fine particle constitutedof silica as the core and coated with gold on the surface and havingparticle diameters ranging from 1 to 100 nm generates heat on absorptionof light of 800 to 1200 nm. The generated heat can be utilized todisconnect the biopolymer compound comprised in the connecting memberand the capping member to separate the capping member from the porousbody.

(Biopolymer compound)

The biopolymer comprised in the connecting member connects physically orchemically the capping member to the porous body. The biopolymer haspreferably a bonding site for bonding to the porous body surface, and abonding site for bonding to the capping member. More preferably at leastone of the bonding sites comprises at least a part of the variableregion of an antibody. The “antibody” in the present invention includesantibodies produced from lymphoid cells of a vertebrate, and proteinswhich have an amino acid sequence derived by deletion, substitution, oraddition of an amino acid constituting the antibody without losing thedesired function as the antibody and by keeping the relation to theoriginal antibody in the constitution and function. The bonding site ofthe connecting member which is capable of bonding to the porous bodysurface and/or the capping member surface may be a fragment, domain, orpart (hereinafter referred to as an “antibody part”) of the antibody.The antibody part which can be a bonding site is exemplified by variableregions V_(H) and V_(L), and composites thereof, the composites beingexemplified by a single chain F_(V) (_(sc)F_(V)) derived from F_(V),V_(H), and V_(L) bonded through a peptide composed of several aminoacids, and a portion thereof.

In the case where gold is contained in at least a portion of the cappingmember surface and the connecting member has a site to bond to the goldportion, the connecting member may comprise a gold-bonding protein. Thegold-bonding protein may comprise an antibody part, and may compriseFab′, (Fab′)², Fd, F_(V), and a part thereof.

In the case where the _(sc)F_(V) is used for constituting the connectingmember, a linker constituted of one or more amino acids is preferablyplaced between V_(H) and V_(L) (no particular arrangement order) formingthe _(SC)F_(V). The residue length of the amino acid linker should bedesigned not to prevent the formation of the necessary constitution forbonding between the V_(H) or V_(L) and the antigen. The length of theamino acid linker is generally 5 to 18 residues: the length of 15residues is most widely employed or investigated.

The aforementioned constitution part of the connecting member can beobtained by a genetic engineering technique.

The bonding site of the biopolymer compound for bonding to the porousbody surface and the bonding site thereof for bonding to the cappingmember may be respectively an antibody part; one site may be an antibodypart and the other site may be a peptide chain constituted of 5 or moreamino acids; or the both sites may be respectively a peptide chainconstituted of 5 or more amino acids. In the description below, theantibody domain constituting the antibody part serving as a bonding siteto the capping member surface is referred to as a first domain, and theantibody domain constituting an antibody part serving as a bonding siteto the porous body surface is referred to as a second domain. FIGS. 4Ato 4D illustrate the bonding sites which are respectively an antibodydomain. In each of the drawings, the reference numeral 1 denotes thefirst domain, and the reference numeral 2 denotes the second domain. InFIG. 4A, the first domain and the second domain are both V_(H); in FIG.4B, the first domain is V_(H) and the second domain is V_(L); in FIG.4C, the first domain is V_(L), and the second domain is V_(H); and inFIG. 4D, the first domain and the second domain are both V_(L). Thebonding sites of the first domain and the second domain are preferablynot complementary. In the connecting member, between the first domainand the second domain, a constituting part other than a polypeptidechain may be provided; the both domains may be joined directly; or theboth domains may be joined with interposition of a polypeptide. Joiningwith a peptide is preferred for simplification in expression of thefunctions and in production process. The interposed peptide may be alinker constituted of one or more amino acids. The linker is preferablyconstituted of 1 to 10 amino acids, more preferably 1 to 5 amino acids.When the linker has a length of 11 to 15 amino acids, the freedom of therelative position of the domains increases to cause complementarybonding between the domains (scFv formation), which can prevent joiningto the capping member and the porous body. Further, the linker may havean embodiment that the linker is designed in a manner that thetwo-dimensional structure allows each of the domains to permit eachother to assume forms capable of easily bonding a target material to bebonded with the domains.

The biopolymer compound having the first and second domains may haveanother domain containing at least a portion of V_(H) and/or at least aportion of V_(L), namely a third domain and/or fourth domain. The thirddomain forms a composite with the first domain, and the fourth domainforms a composite with the second domain. For example, when the firstdomain is V_(H), the third domain is V_(L) capable of forming F_(V) withthe first domain, and the resulting composite forms a bonding site incombination of the first domain and the third domain for bonding to acapping member surface. Similarly, when the second domain is V_(L), thefourth domain is V_(H) capable of forming F_(V) with the second domain,and the resulting composite forms a bonding site in combination of thesecond domain and the fourth domain for bonding to a porous bodysurface.

FIGS. 5, 6, 7, 11, 12, and 13 illustrate schematically the state of thecomposite of the first domain with the third domain. FIGS. 8, 9, 10, 11,12, and 13 illustrate schematically the state of the composite of thesecond domain with the fourth domain. The formation of the composite canstabilize the constitution to retard drop of the function byconstitution change. Such a composite forms preferably a bonding site incombination for bonding to the capping member surface or the porous bodysurface. The formation of the bonding site in combination of the domainscan be effective in improving the bonding properties such as increase ofthe bonding rate, retardation of the dissociation rate, and so forth. Insuch a manner, the constitution can be selected for bonding the domainand domain composite to the porous body surface and the capping membersurface.

The constitution shown schematically in FIG. 7 can be employed in whicha polypeptide chain comprises the first domain and the second domain andin which the third domain forms a composite with the first domain. Withthis constitution, F_(V) or an F_(V)-like composite formed from thefirst domain and the third domain bonds to the capping member surface,and the bonding of the two second domains to the porous body serves asan anchor biting the porous body. Similarly, the constitution shownschematically in FIG. 10 can be employed which is constituted of apolypeptide chain comprising the first domain and in which the seconddomain and the fourth domain forms composite with the first domain. Withthis constitution, F_(V) or an F_(V)-like composite formed from thesecond domain and the fourth domain bonds to the porous body surface,and the bonding of the two first domains to the capping member serves asan anchor biting the capping member body.

The pair of the domains forming the composite may be respectively anindependent polypeptide chain, or may be linked together directly by alinker. The linker for linking the domains of the composite havingbonding sites for bonding to the capping member and the porous body haspreferably a chain of 1 to 10 amino acids, more preferably 1 to 5 aminoacids by the same reason as the aforementioned linker for connecting thedomain having a bonding site to the capping member and the domain havinga bonding site to the porous body, but may be longer if necessary. Forexample, in the case of FIG. 13, the first and second domains, and thethird and fourth domains may be respectively linked by 1 to 5 aminoacids, and the second and third domains may be linked by 15 to 25 aminoacids. FIG. 6 illustrates schematically a polypeptide chain in which thefirst and third domains are linked by a linker. FIGS. 9 and 13illustrate schematically a polypeptide in which the second and fourthdomains are linked by a linker. In FIGS. 6, 9, and 13, the first andsecond domains are linked by a linker, and further in FIG. 13, the thirdand fourth domains are linked by a linker. Therefore, sequences ofpolypeptide chains are formed: second-first-third domains in FIG. 6,first-second-fourth domains in FIG. 9, and first-second-fourth-thirddomains in FIG. 13. The arrangement of the domains in thesingle-stranded polypeptide can be selected suitably for intendedproperties such as properties for bonding to gold or a target, andlong-term stability of the gold-bonding protein.

Further, the biopolymer compound constituted of an antibody fragment andhaving the aforementioned domain composite for forming a bonding site(e.g., the first and third domains, and the second and forth domains)can be modified genetically at a portion which does not affect greatlythe intended bonding property. For example, a disulfide bond is formedat the bonding interface between the domains by introducing a cysteineresidue at the interface between the domain composite (e.g., the firstand third domain, or the second and fourth domains). Otherwise, twocysteine moieties are introduced in the linker to promote formation of adomain composite for forming the same bonding sites or for improving thestability.

The variable region of the antibody for use as the connecting member haspreferably at least one of the amino acid sequences: SEQ ID NO:1 to SEQID No:57. The variable region may have also an amino acid sequence orsequences selected from the amino acid groups obtained by deletion,substitution, or addition of one or more amino acids to or from any ofthe above 57 amino acid sequences, provided that the connecting membercan function as intended. The amino acid sequences of the variableregion of the antibody having the sequence of SEQ ID NO:1 to SEQ IDNO:57 are exemplified by the sequences SEQ ID NO:58 to SEQ ID NO:77. Inthis connection, SEQ ID NO:58 to SEQ ID NO:77 are denoted by thefollowing names in the present specification in a manner as “58: 7s1”which means SEQ ID NO:58 is denoted by a name 7s1: 58: 7s1, 59: 7s2, 60:7s4, 61: 7s7, 62: 7s8, 63: 7p2, 64: 7p3, 65: 7p4, 66: 7p7, 67: 7p8, 68:10s1, 69: 10s2, 70: 10s3, 71: 10s4, 72: 10s5, 73: 10p1, 74: 10p2, 75:No.4, 76: No.7, and 77: No.10.

A biopolymer compound like this can be prepared genetically as aprotein. The biopolymer compound can be obtained as a fused protein byintroduction of a nucleic acid coding for the biopolymer compound to anexpression vector, expression, and purification. The protein may beprepared as a single strand polypeptide chain. In formation of a singlestranded polypeptide, for example, a nucleic acid for coding for apeptide affinitive to a substrate is introduced to a 5′-terminal (or3′-terminal) of a nucleic acid for coding a variable region of anantibody to obtain a nucleic acid for coding for a protein fused with asubstrate-affinitive peptide at an N-terminal (or C-terminal) of thevariable region. In this case, it is also possible to insert a nucleicacid sequence which codes a amino acid sequence of the above linkerbetween the nucleic acid sequence coding the antibody fragment and thenucleic acid sequence coding the affinitive peptide chain. Further, thebiopolymer compound may be constituted of a composite of a pluralpolypeptide chains. The constitution of the protein is not limitedprovided that the necessary function of the connecting member is notimpaired.

In the case where the connecting member of the present invention has anantibody fragment and an affinitive peptide as the bonding sitesrespectively to the porous body and the capping member. For example, aprotein can be formed by fusion of the above affinitive peptide to avariable region of an antibody, or an N-terminal or C-terminal of acomposite of the antibody. The peptide chain affinitive to the cappingmember can be selected in consideration of the bonding ability by designby calculation and screening by a peptide presentation method such as aknown phage display method. Otherwise, the selection can be made bypreparing a candidate library with reference to known amino acidsequences (Nature Materials, Vol.1, 2, pp577, 2003) and screening them.For the porous body composed of a silicon oxide type material,especially SBA-15, suitable are SEQ ID NO:80 to SEQ ID NO:81.

In the same manner as above, in a combination of _(SC)F_(V) moietiesforming a bonding site (e.g., the first domain and the third domain; orthe second domain and the fourth domain mentioned above), a part thereofwhich does not affect the intended bonding properties may be geneticallymodified. For example, the modification can be made by formation of adisulfide linkage at the joining interface between _(SC)F_(V) moieties:a cysteine residue is introduced to the bonding interface between theportions of _(SC)F_(V) (e.g., the first domain and the third domain; orthe second domain and the fourth domain); or two cysteine residues areintroduced to the linker to assist formation of _(SC)F_(V) having thesame bonding site for stabilization.

The protein-expressing vector can be designed and constructed byincorporating construction for expressing gene for coding for abiopolymer compound like a known promoter corresponding to a selectedhost cells. When Escherichia coli is used as the host cells, thedecomposition by a protease can be retarded by removing a protein or itsconstituent, a foreign genetic product, quickly to the outside of thecell. If the foreign genetic product is toxic to the bacteria, it isknown that the adverse effect can be minimized by secreting it to theoutside of the cell. Usually, most of the proteins secreted through acell membrane or inside membrane have a signal peptide at the N-terminalof the precursor thereof, and is cleaved by a signal peptidase in thesecretion process to become a maturation protein. Most of the signalpeptides have a basic amino acid, a hydrophobic amino acid, or a site ofcleavage by a signal peptidase at the N-terminal thereof.

The objective protein can be secreted and expressed by placing a nucleicacid for coding for a known signal peptide typified by pelB (a signalpeptide) at the 5′ side of a nucleic acid for coding for the protein.

In the case where a biopolymer compound is formed as a composite ofplural polypeptide chains, the polypeptide may be prepared respectivelyby separate vectors, but the plural polypeptide chains can beconstructed by one vector. In this case, the secretion can be promotedby placing a nucleic acid for coding for pelB at the 5′ side of thenucleic acid for coding for the domains or polypeptide chain. Further,for expression of a polypeptide chain having one or more domains, thesecretion can be promoted by placing a nucleic acid for coding for pelBat the 5′ terminal of the-polypeptide chain in the same manner as above.The fused protein having a signal peptide at the N-terminal can bepurified from a periplasma fraction and a culture supernatant.

For simplicity of the operation of purification of the expressedprotein, a tag for purification can be provided genetically at theN-terminal or C-terminal of an antibody molecule, or a polypeptide chainformed by combining independent antibody fragments or plural antibodyfragments. For example, the tag for purification is exemplified by ahistidine tag composed of 6-resudue sequence of histidine (hereinafterreferred to as His×6), and a gluthathion-linking site of aglutathione-S-transferase (GST). The tag can be introduced by insertionof a nucleic acid for coding for the purification to 5′- or 3′-terminalof a nucleic acid for coding for gold-bonding protein in an expressionvector into an intended position, by using a commercial vector forpurification tag introduction, or by a like method.

An example of the process of the biopolymer by employing theaforementioned expression vector is described below.

The protein which is a biopolymer compound, or a polypeptide which is aconstituting element of the protein is synthesized in a known host cellfor protein expression by transforming the aforementionedprotein-expressing vector designed for the host cell and using theprotein synthesis system in the host cell. Then the intended protein isobtained by separating and purifying the cell liquid or a culturesupernatant. For example, with Escherichia coli as the host cell, theprotein can be allowed to secrete outside the cell by placing a nucleicacid for coding for a known signal peptide such as pelB at the 5′ sideof the nucleic acid.

Plural polypeptide chains having gold-bonding protein as a componentelement can be coded for in one expression vector. For this purpose, anucleic acid for coding for pelB is placed at the 5′ side of the nucleicacid for coding for the respective polypeptide chains as theconstitution elements to promote the expressed protein outside the cell.

The gold-bonding protein having a signal peptide fused at the N-terminalcan be purified from a periplasma fraction and a culture supernatant.For the purification, the protein component is concentrated from thefraction by ammonium sulfate or the like, suspended again in a suitablebuffer solution, and purified: for example, by a nickel chelate columnwhen a His tag is employed as the purification tag, and by aglutathione-immobilizing column when a GST is employed as thepurification tag.

The gold-bonding protein expressed in a bacteria cell can be obtained asinsoluble granules. The granules can be isolated by separating thebacterial mass from the liquid culture, crushing the bacterial mass by aFrench press or ultrasonic wave, and centrifuging the crushed cellliquid. The obtained insoluble granule fraction is solubilized by usinga buffer solution containing a known modifying agent containing urea, ora guanidine hydrochloride salt, and purified by use of a purifyingcolumn under the modification conditions as above. The obtained columneluate fraction is subjected to a refolding treatment for removal of themodifier and reconstruction of the active structure. The refoldingtreatment may be conducted by any known method, including a stepwisedialysis, and dilution corresponding to the intended protein.

The domains and polypeptide chains of the gold-bonding protein can beexpressed in the same host cell, or can be expressed in separate hostcells and brought together to form a composite.

The vector containing the nucleic acid for coding for the gold-bondingprotein is known to be capable of expressing the intended protein invitro with a liquid cell extract. Suitable cells therefor includeEscherichia coli, wheat germ, and rabbit retiform erythrocyte. However,since the protein synthesis from the cell-free liquid extract isconducted under reduction conditions, the conventional refoldingtreatment is preferred for formation of the disulfide linkage in theantibody fragment.

One of the bonding sites of the biopolymer compound for bonding to theporous body surface or to the capping member surface may be modified byintroduction of a chemical modifying group into the biopolymer compound.For example, for a capping member having gold bared at least on aportion of the surface thereof, a biopolymer compound of the presentinvention can be prepared by introducing a group having an SH group atthe terminal portion other than the bonding site for bonding to theporous body surface. On the other hand, to a biopolymer having a sitefor bonding to gold, a functional group having a silanol group or analkoxysilane group may be introduced.

The bonding site of the connecting member for bonding to the porous bodycan be formed to have a peptide portion capable of bonding to the porousbody. For a porous body having at least one of metals and metal oxideson at least a part of the surface, the connecting member can beconstituted such that the bonding site of the connecting member connectswith the portion containing at least one of the metals and metal oxides.Otherwise, for the porous body having silicon oxide on at least a partof the surface, the bonding site of the connecting member may beconstituted so as to bond to the portion containing at least one ofmetals and metal oxides. The bonding site for bonding to the porous bodycan be a peptide having two or more of amino acid sequences selectedform the group of amino acid sequences (1) SEQ ID NO:80, and amino acidsequences derived from (2) SEQ ID NO:81 by deletion, substitution, oraddition of one or more amino acids.

(Releasable Substance)

The substance to be held by the structure of the present invention isselected to meet the use of the structure, being selected from variouscompounds and materials such as water-soluble drugs and fat-solubledrugs.

(Controlled Release of Compound)

The retention and release of a substance in or from the structure of thepresent invention is controlled by an external stimulation, namely byloading of an external signal.

The external signal includes light, magnetism, and electricity. For DDSapplication, the signal is preferably harmless to a living bodyreceiving the released compound. Therefore the light and the magnetismare preferred. The wavelength of the light is selected in considerationof influences on the capping member and the compound-receiving body. Forexample, a fine particle (particle size: 1-100 nm) having a core(particle size: 5-50 nm) coated with gold (coating thickness: 1-6 nm)generates heat by absorbing light of 800-1200 nm which is almostharmless to tissues and cells of animal bodies: The generated heat canbreak the bonding between the biopolymer compound and the capping memberand disconnect the capping member from the porous body to release thecompound to the outside of the structure.

A capping member which is formed from fine particulate gold is known togenerate heat by application of a high frequency wave of 10 MHz to 2 GHzin a region from short wave to microwave. Such a change of a thermalproperty of the capping member can be utilized for disconnecting thecapping member from the structure for the controlled compound release ofthe present invention. The light irradiation time is selected dependingon the wavelength of the irradiated light.

The signal inputting apparatus for the release controlling means of thepresent invention is not limited provided that the apparatus is capableof changing the temperature of the periphery of the capping memberconnected to the porous body holding the substance of the presentinvention. For example, the apparatus may be a lamp for projecting alight beam of a wave length of 800 to 1200 nm, or may be a YAG laser, orthe like, or may be any type of microwave-generating apparatus.

FIG. 1 illustrates schematically a process of release or diffusion ofthe drug. Drug 12 is held inside pore 11 of a porous body (13).Separately, a metal-coated fine silica particle 14 is prepared bycoating a fine silica particle of 30 μm diameter with gold in athickness of t=3 nm. Onto the surface of the fine silica particle,gold-bonding F_(V) 15 fused with a silica-affinitive peptide is bonded.Thereby F_(V) is bonded to the surface of the porous body constitutingthe opening of pore 11, and the fine particle 14 bonded with F_(V) isheld at the opening of the pore to close the pore (16). When the porousbody is placed in a photomagnetic field, the fine particle absorbsenergy (17) to generate heat. Consequently, the bonding of F_(V) isbroken and the fine particle is removed from the opening to release drug12 from the pore outside by diffusion (18). Incidentally, drug 12 may bekept near the opening, not inside the pore provided that the drug isprevented from diffusion.

The structure of the present invention gives further second to seventheffects shown below.

As second effect, a porous body surface and a capping member surface canbe specifically connected together through an organic compound as aconnecting member including a biopolymer compound having capability ofbonding to the porous body surface and to the capping member surface,especially an antibody, its fraction, or a peptide. Thereby, an adverseeffect of bond formation between the connecting member and a substancenot to be bonded thereto such as the releasable substance or a likeinteraction can be prevented. Therefore, higher specificity inrecognition can be expected by use of a biopolymer compound which has avariable region of an antibody capable of bonding to the porous bodysurface and/or a variable region of an antibody capable of bonding tothe capping member surface.

As a third effect, the porous body can be made stable in an environmentof a body fluid such as blood by constituting the porous body at leastpartly from a metal or a metal oxide. Further, biocompatibility of thestructure can be increased by forming the porous body from silicon oxideand the capping member from gold.

As a fourth effect, a releasable substance can be surely released fromthe porous body at an intended time and a site by designing thestructure such that the capping member is disconnected by input ofexternal stimulation (releasing signal) and the substance kept in thestructure is released therefrom in accordance with the inputted signal.

As a fifth effect, the substance can be released by removal of thecapping member by a slight temperature change that does not affectadversely the porous body constituting the structure, the substance keptin the porous body, and a treated portion of an objective patient, sincea biopolymer compound is employed for physical or chemical connection ofthe porous body surface and the capping member surface.

As a sixth effect, the signal for inducing the release of the substanceis inputted by at least one of light and a magnetic field change tocause temperature change at least on the surface of the capping member.The light and the magnetic field can be selected to be actuallyharmless, when applied, to treated animal bodies and human bodies.Therefore, an expensive equipment or apparatus is not necessary for thecontrolled release of the substance, and the application field will bewidened.

As the seventh effect, the gold placed on at least a part of the cappingmember surface enables selection of the light or the magnetism changeregion to be harmless to the living body, and stable retention of thecapping member surface to give long-term stability of the function ofthe controlled-release.

EXAMPLES

In Examples below, a structure is formed from mesoporous silica (SBA-15)as the porous body, gold-coated silica beads (gold layer: 1 nm, silica:10 nm diameter) as the capping member, and a fused protein as biopolymercompound for connection prepared by fusion of an SBA-15-affinitivepeptide to a C-terminal of a gold-bonding _(SC)F_(V).

Example 1 Preparation of Porous Body

Mesoporous silica (SBA-15) is prepared by the procedure below.

A silica reaction solution is prepared from 4 g of a poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymerconstituted of 20 units of ethylene oxide, 70 units of propylene oxide,and 20 units of ethylene oxide (hereinafter referred to asEO₂₀-PO₇₀-EO₂₀); 0.041 mol of tetraethoxysilane (TEOS); 0.24 mol of HCl;and 6.67 mol of H₂O.

This silica reaction solution is allowed to react at 35° C. for 20hours, and at 80° C. for 48 hours. The solution is further heated at500° C. for 6 hours to burn the contained block copolymer resinEO₂₀-PO₇₀-EO₂₀ to obtain a porous silica.

The resulting porous silica has pores having an average pore diameter of7.9 nm with an average silica wall thickness between the pores of 3 nm.

Example 2 Preparation of Fusion Protein of Gold-Bonding _(SC)F_(V) andPeptide Affinitive to SBA-15

A protein formed by fusion of a peptide of SEQ ID NO:80 affinitive toSBA-15 to the C-terminal of a gold-_(SC)F_(V) through the steps below.

(1) Preparation of Expression Vector

A plurality of sets of pET-15b (Novagen Co.) were modified by cuttingthe multiclonibng site thereof with NheI/SacII and NotI/SacIIrespectively to prepare two sets of pUT-XX as shown in FIGS. 2A and 2B.Next, VL (clone name: VL No.7, SEQ ID No.76 and VH (clone name: 7s4, SEQID NO:60), which will become constituents of a gold-bonding scFv, wereinserted into the vectors pUT-XX, respectively. The resultant vectorsare referred to as pUT-VL No. 7 and pUT-7s4, respectively. Then anexpression vector pUT-_(SC)F_(V) was prepared as below in which theVL-coding gene, a linker (SEQ ID NO:94×3), the VH-coding gene, aSBA-15-affinitive peptide (hereinafter may be referred to as “Si tag” asshown in FIGS. 3A to 3C) and a His×6 (hereinafter referred to as a Histag) are translated continuously and expresses it as a fusion protein(FIGS. 3A to 3C).

PCR is conducted with the above pUT-7s4 as a template and by using theprimers below:

SiscF_(v)-B (SEQ ID NO:78) SiscF_(v)-F (SEQ ID NO:79)The PCR is conducted by using a commercial PCR kit (Takara Bio K. K.,LA-Taq Kit) according to a protocol recommended by the supplier.

The obtained PCR product is subjected to 2%-agarose electrophoresis, androughly purified by a gel of a gel extraction kit (Promega Co.) toobtain a PCR fraction of about 400 bp. The product is confirmed to havethe intended base sequence by sequencing. pUT-VL No.7 and the PCRfragment obtained in the above PCR are cut by NotI/SacII. The productsare subjected to agarose electrophoresis, and the intended fragments arepurified at the vector side and the insert side.

The obtained purified nucleic acid fragment is mixed at a ratio ofVector:Insert=1:5 and the mixture is subjected to a ligation reaction inthe same manner as in Example 1.

With the above ligation reaction liquid, JM109 competent cell 40 μL istransformed. The transformation is conducted by heat shock bytemperature change from an ice temperature to 42° C. for 90 sec to anice temperature. To the above BL21 solution after transformation by heatshock, 750 μL of an LB culture is added and the mixture is cultivated at37° C. for one hour by shaking. Then the culture is centrifuged at 6000rpm for 5 minutes. A 650 μL portion of the supernatant liquid of theculture is discarded. The remaining supernatant liquid and theprecipitated cell fraction are stirred, spread on an LB/amp. plate, andleft standing at 37° C. overnight.

From the plate, colonies are picked out at random, and are cultivated in3 mL of an LB/amp. liquid culture by shaking. Therefrom, a plasmid isextracted by a commercial MiniPrep kit (Promega Co.) according to amethod recommended by the supplier. The obtained plasmid is cut byNotI/SacII. The product is subjected to agarose electrophoresis toconfirm the insertion of the intended gene fraction. This plasmid isreferred to a pUT-_(SC)F_(V)Sp.

With the plasmid pUT-_(SC)F_(V)Sp obtained in the above operation, BL21(DE3) competent cell 40 μL is transformed. The transformation isconducted by heat shock by temperature change from an ice temperature to42° C. for 90 sec and to an ice temperature.

To the above BL21 solution after transformation by heat shock, 750 μL ofan LB culture is added and the mixture is cultivated at 37° C. for onehour by shaking. Then the culture is centrifuged at 6000 rpm for 5minutes. A 650 μL portion of the supernatant liquid of the culture isdiscarded. The remaining supernatant liquid and the precipitated cellfraction are stirred, spread on an LB/amp. plate, and left standing at37° C. overnight.

(2) Preliminary Cultivation

From the plate, colonies are picked out at random, and are cultivated in3.0 mL of an LB/amp. liquid culture at 28° C. overnight by shaking.

(3) Main Cultivation

The above preliminary cultivation liquid is inoculated to 750 mL of a2×YT culture, and cultivation is continued at 28° C. At the time whenthe OD600 exceeds a level of 0.8, IPTG is added thereto to a finalconcentration of 1 mM. The mixture is further cultivated at 28° C.overnight.

(4) Purification

Through Steps (A) to (E) below, the intended polypeptide chain isrecovered from the insoluble granule fraction, and is purified.

(A) Recovery of Insoluble Granules

The culture liquid obtained in the above Step (3) is centrifuged at 6000rpm for 30 minutes to obtain a bacterial mass fraction as a precipitate.The bacterial mass is suspended in 15 mL of a tris solution (20 mMtris/500 mM NaCl) on an ice bath. The liquid suspension is crushed by aFrench press to obtain a crushed cell liquid. The crushed cell liquid iscentrifuged at 12000 rpm for 15 minutes. The supernatant liquid isremoved to obtain an insoluble granule fraction as a precipitate.

(B) Solubilization of Insoluble Granule Fraction

The insoluble fraction obtained in Step (A) is immersed in 10 mL of 6Mguanidine hydrochloride/tris solution overnight. The solution iscentrifuged at 12000 rpm for 10 minutes to obtain a solubilized solutionas the supernatant liquid.

(C) Metal Chelate Column

His-Bind (Novagen Co.) is used as a stationary phase of a metal chelatecolumn. The column preparation, sample loading, and washing areconducted according to the method recommended by the supplier at roomtemperature (20° C.). The intended His-tagged fusion polypeptide iseluted by a 60 mM imidazole/tris solution. The SDS-PAGE (acrylamide 15%)measurement of the eluate shows a single band, whereby the polypeptideis confirmed to be purified.

(D) Dialysis

The above eluate is dialyzed by use of a 6M guanidine hydrochloride/trissolution as the external liquid at 4° C. to remove the imidazole fromthe eluate and to obtain the respective polypeptide chain solution.

(E) Refolding

In the same manner as above, the solutions of the polypeptide chains of_(SC)F_(V)-Sp, a fusion product of the gold-bonding F_(V) and the abovepeptide, are respectively refolded with simultaneous removal ofguanidine hydrochloride by dialysis at 4° C. through Steps (a) to (g)below.

-   (a) Polypeptide chain samples are prepared with a 6M guanidine    hydrochloride/tris solution at a concentration of 7.5 μM (volume 10    mL after dilution) by measuring the molar extinction coefficient and    the value ΔO.D. (280-320 nm) of the respective polypeptide chains.    To the sample solutions, β-mercaptoethanol (reducing agent) is added    to a final concentration of 375 μM (50 times the protein    concentration) and reducing reaction is allowed to proceed at room    temperature in the dark for 4 hours. This sample solutions are put    into a dialysis bag (MWCO: 14,000) for dialysis.-   (b) The samples for dialysis are immersed in a 6M guanidine    hydrochloride/tris solution as the external solution for dialysis.    Dialysis is conducted with gentle stirring for 6 hours.-   (c) The concentration of the external solution is lowered stepwise    to 3M, and 2M. The dialysis is conducted at each of the external    solution concentrations for six hours.-   (d) To a tris solution, are added an oxidation type glutathione    (GS-SG) to a final concentration of 375 μM, and L-Arg to a final    concentration of 0.4 M. This tris solution is added to the 2M    dialysis external solution of the above Step (c) to bring the    guanidine hydrochloride concentration to 1 M, and the pH of the    mixture solution is adjusted to pH 8.0 (4° C.) by NaOH. With this    solution, the dialysis is continued with gentle stirring for 12    hours.-   (e) In the same manner as in the above Step (d), an L-Arg-containing    tris solution containing 0.5 M guanidine hydrochloride is prepared,    and the dialysis is continued with this solution for further 12    hours.-   (f) Finally the dialysis is conducted in the tris solution for 12    hours.-   (g) After-the dialysis, the agglomerate and the supernatant are    separated by centrifugation at 10000 rpm for-about 20 minutes. The    solution obtained above is subjected to SPR measurement by replacing    the external solution to a phosphate buffer (hereinafter referred to    as PBS). Thereby the property of bonding to gold is confirmed.

Example 3 Preparation of Structure

-   (1) A 200 mg portion of SBA-15 prepared in Example 1 is immersed in    a 3 μM ATP/phosphate buffer solution; PBS (pH 7.4) overnight.-   (2) A fine particulate gold (20 nm, produced by Tanaka Kikinzoku K.    K., 0.15 mmol) is suspended in 0.01 mmol ATP/PBS.-   (3) The 1.5 μM _(SC)F_(V)/PBS fused with silica-affinitive peptide    prepared in Example 2 is mixed with the immersion-treated SBA-15 of    Step (1) and the suspension of Step (2), and the mixture is stirred    for 24 hours.-   (4) Then the suspension is centrifuged at 12000 rpm for 5 minutes to    remove the supernatant to obtain a precipitate. This precipitate is    vacuum-dried to obtain a structure.

Example 4 Controlled Release of Compound from Structure-1

-   (1) A 20 mg portion of the structure obtained in Example 3 is    suspended in a PBS (pH 7.4) and is dispersed in the solution by    application of an ultrasonic wave. This operation is repeated three    times to wash off the adsorbed ATP.-   (2) The above structure is kept standing in a state of suspension    for 12 hours. At 0, 4, 8, and 12 hours of the standing, a portion of    the solution is taken out. The respective portions are subjected to    measurement by HPLC (C18, reversed phase column, detection    wavelength: 275 nm). The amount of ATP is confirmed to decrease with    time.-   (3) Then a YAG laser light (1064 nm, 164 mJ/pulse, 7 nsec, 10 Hz) is    projected thereto for one hour.-   (4) During the laser irradiation, a sample is taken out from the    solution in every 10 minutes and is subjected to HPLC analysis.    Thereby ATP is confirmed to be released with time.

Example 5 Controlled Release of Compound from Structure-2

-   (1) A 20 mg portion of the structure obtained in Example 3 is    suspended in a PBS (pH 7.4) and is dispersed in the solution by    application of an ultrasonic wave. This operation is repeated three    times to wash off the adsorbed ATP.-   (2) The above structure is kept standing in a state of suspension    for 12 hours. At 0, 4, 8, and 12 hours of the standing, a portion of    the solution is taken out. The respective portions are subjected to    measurement by HPLC (C18, reversed phase column, detection    wavelength: 275 nm). The amount of ATP is confirmed to decrease with    time.-   (3) Then a light beam of 0.5 GHz is projected thereto intermittently    five times at a cycle of 10 seconds of projection and 50 seconds of    interruption by a synthesized signal-generation apparatus (HP Co.).-   (4) After the signal projection, a sample is taken out from the    solution and is subjected to HPLC analysis. The amount of the ATP    increases in comparison with an amount of ATP in the solution in    Step (2). This shows that the ATP is released by the light signal.

Example 6

A protein is prepared, using a VH (VH clone name: A14P-7s4, SEQ ID NO:83) corresponding to a base sequence represented by SEQ ID NO:82, whichVH was introduced by replacing the fourteen amino acid residue, alanine,of VH in the gold-bonding scF_(V)-SBA-15-affinitive peptide employed inExample 2 with proline.

Preparation of Expression Plasmid:

A variation is introduced to the intended position by using, as atemplate, pUT-_(SC)F_(V)Sp obtained in Example 2. The variation isintroduced by means of QuickChange Kit (Stratagen Co.) according to amethod recommended by the supplier. The primers below are employed.

A14P-f [SEQ ID NO:84] A14P-r [SEQ ID NO:85]By sequencing, the obtained plasmid is confirmed to have the intendedDNA coding the amino acid sequence represented by the SEQ ID NO:80.

By use of the plasmid pUT-_(SC)F_(V)2Sp, BL21 (DE3) competent cell 40 μLis transformed by heat shock by temperature change from an icetemperature to 42° C. for 90 seconds and to an ice temperature.

To the above BL21 solution having been transformed by the heat shock,750 μL of an LB culture is added. The mixture is cultivated at 37° C.for one hour by shaking.

The culture is centrifuged at 6000 rpm for 5 minutes. A 650 μL portionof the culture supernatant is discarded, and the remaining supernatantand the precipitated cell fraction are stirred. This mixture is spreadon an LB/amp. plate, and is kept standing at 37° C. overnight.

Then, the intended protein is obtained in the same manner as in Steps(2) to (4) in Example 2. A structure is prepared by use of the obtainedprotein in the same manner as in Example 3.

The evaluation is made in /the same manner as in example 3 and it isconfirmed that the ATP is released in the same manner as in Example 4.

Example 7

A protein is prepared which corresponds to VH (VH clone name: PFER-7s4,SEQ ID NO:87) represented by SEQ ID NO:86. The protein has constitutionof the VH (VH clone name: A14P) of the gold-bonding_(SC)F_(V)-SBA-15-affinitive peptide employed in Example 6 in which thevaline at 34^(th) position is changed to phenylalanine and the glutamineat 44^(th) position is changed to glutamic acid, and the leucine at45^(th) position is changed to arginine.

Preparation of Expression Plasmid:

Variations are introduced to the intended positions by using, as atemplate, pUT-_(SC)F_(V)2Sp obtained in Example 6. The variations areintroduced by means of QuickChange Kit (Stratagen Co.) according to amethod recommended by the supplier. The intended plasmid is obtained bythree operations. The primers below are employed.

PCR Primer for the First Variation Introduction

V37F-f [SEQ ID NO:88] V37F-r [SEQ ID NO:89]PCR Primer for the Second Variation Introduction

G44E-f [SEQ ID NO:90] G44E-r [SEQ ID NO:91]PCR Primer for the Third Variation Introduction

L45F-f [SEQ ID NO:92] L45F-r [SEQ ID NO:93]By sequencing, the obtained plasmid is confirmed to have the intendedDNA represented by the SEQ ID NO:82 inserted therein.

By use of the plasmid pUT-_(SC)F_(V)2Sp obtained by the above operation,BL21 (DE3) competent cell 40 μL is transformed by heat shock bytemperature change from an ice temperature to 42° C. for 90 seconds andto an ice temperature.

To the above BL21 solution having been transformed by the heat shock,750 μL of an LB culture is added. The mixture is cultivated at 37° C.for one hour by shaking.

The culture is centrifuged at 6000 rpm for 5 minutes. A 650 μL portionof the culture supernatant is discarded, and the remaining supernatantand the precipitated cell fraction are stirred. This mixture is spreadon an LB/amp. plate, and is kept standing at 37° C. overnight.

Then, the intended protein is obtained in the same manner as in Steps(2) to (4) in Example 2. A structure is prepared by use of the obtainedprotein in the same manner as in Example 3.

The evaluation is made in the same manner as in example 3 and it isconfirmed that the ATP is released in the same manner as in Example 4.

INDUSTRIAL APPLICABILITY

The present invention provides a structure comprising at least a porousbody holding one or more compounds, and comprising (1) a capping memberfor keeping the compound in a pore or a periphery thereof, and (2) amaterial for connecting the capping member and the surface of the porousbody physically or chemically, the material comprising an organiccompound having a biopolymer as at least a part thereof. According tothe present invention, a structure can be provided which is capable ofholding a compound stably in the porous body. The present inventionprovides also a means for controlling the release of the compound fromthe structure. According to the present invention, the compound held bythe structure can be released at a desired timing from the structure.

The present invention enables supply of an intended substance suitablyin a chemical reaction. By application of the present invention to drugdelivery systems, the intended substance can be released by applying asignal from the outside to a diseased portion only, whereby the amountof a drug dosed to a patient can be minimized to decrease adverseeffects such as a side effect.

SEQUENCE LISTING FREE TEXT <210> 78 <223> primer for PCR <210> 79 <223>primer for PCR <210> 80 <223> sequence for deriving amino acid sequencefor binding to porous member <210> 81 <223> sequence for deriving aminoacid sequence for binding to porous member <210> 82 <223> Variableregion of heavy chain A14P-7s4 coding DNA <210> 83 <223> Variable regionof heavy chain A14P-7s4 <210> 84 <223> primer for PCR <210> 85 <223>primer for PCR <210> 86 <223> Variable region of heavy chain PFER-7s4coding DNA <210> 87 <223> Variable region of heavy chain PFER-7s4 <210>88 <223> primer for PCR <211> 33 <223> primer for PCR <210> 90 <223>primer for PCR <210> 91 <223> primer for PCR <210> 92 <223> primer forPCR <210> 93 <223> primer for PCR <210> 94 <223> linker

This application claims priority from Japanese Patent Application No.2004-108329 filed Mar. 31, 2004, which is hereby incorporated byreference herein.

1. A structure for holding a compound inside of the structure,comprising: a porous body having pores with a diameter ranging from 1 nmto 10 μm which comprises silicon oxide; a capping member for covering atleast a part of an opening of at least one of the pores; and aconnecting member for connecting the capping member to a surface of theporous body, wherein, the capping member is a particle at least thesurface of which particle comprises gold, and the diameter of thecapping member is greater than the size of the opening of the pores ofthe porous body, and wherein, the connecting member comprises at leastone variable region of an antibody which region comprises the amino acidsequence of SEQ ID NO: 1 and binds to the gold.
 2. The structureaccording to claim 1, wherein the at least one variable region furthercomprising one or more amino acid sequences selected from the groupconsisting of the amino acid sequences of SEQ ID NOS: 2 to
 57. 3. Thestructure according to claim 1, wherein the diameter of the cappingmember ranges from 2 nm to 50 μm.